This project focuses on HIV-1 replication in cultured cells and in vivo to develop understandings of viral pathogenesis and the cellular factors that influence viral gene expression. Important scientific advances that we have achieved in the 2008 to 2009 period include the following. 1) We have studied a series of ring expanded nucleoside (REN) analogues and have identified two which inhibit the ATP dependent activity of human RNA helicase DDX3 and abrograte HIV-1 replication in cultured human cells. 2) We have shown that the prolyl isomerase Pin1 protein modulates the expression of an HIV-1 restriction factor APOBEC 3G. We found that Pin1 is an APOBEC3G-interacting protein that acts to negatively regulated APOBEC3G's inhibition of HIV-1 replication. 3) We have characterized the microRNA expression pattern in peripheral blood mononuclear cells from 36 HIV-1 seropositive individuals and 12 normal subjects. We found that HIV-1 infection results in specific signature profiles of microRNA expression in HIV-1-positive individuals. Some of these expression patterns appear to correlate with T-cell hyperactivation. 4) To identify cellular proteins that contribute to HIV-1 replication that can be chronically silenced without significant cytotoxicity, we employed a shRNA library that targets 54,509 human transcripts. Using this approach, we identified 252 individual Jurkat mRNAs that contribute to HIV-1 replication. 5) We have performed linker-ligated cloning followed by high-throughput pyrosequencing of HIV-1 infected T cells. We observed that many discrete small HIV-1 non-coding RNAs were identified by this type of high sensitivity "deep" sequencing.